The emerging concept of the neurovascular unit emphasizes that cell-cell interactions between vascular and parenchymal compartments mediate an integrative response to injury. Thus, endothelial cell dysfunction may be an upstream trigger for brain damage after stroke. For this R21 Project, we propose to investigate the Proteomic Profiles of Human Brain Endothelial Cells in Hypoxia, to broadly characterize the role of soluble and cytosolic mediators in this in vitro stroke model. Building on our encouraging preliminary findings of novel proteinases and substrates found in oxidative stress, we hypothesize that: In comparison to controls, human endothelial cell cultures exposed to hypoxic insult will have consistent and identifiable soluble-protein and substrate expression profiles, indicating relative up- or down-regulation of both known and unknown factors important in cell-cell and cell-matrix signaling. We propose a three-part study in human brain endothelial cell culture media and lysates, to investigate the mechanism of hypoxia-reoxygenation. Specific Aim 1: Using validated techniques of quantitative proteomics technology, examine the protein expression profile of human endothelial cell culture media pre and post hypoxia-reoxygenation. Specific Aim 2: Examine the down-stream substrates repertoire of human endothelial cell culture media pre and post hypoxia-reoxygenation, in order to obtain relative quantification of the substrate repertoire characteristic of hypoxic injury. Secondary Aims: Extend Aim 1 and 2 to study cell lysates. Through quantitative proteomic analysis of hypoxia-reoxygenation in human brain endothelial cells, an important component of the neurovascular unit, we hope to provide a screening tool for the discovery of novel proteins in ischemic models, and provide venues for the discovery of new therapeutic targets.